Project Summary CLL is the most common leukemia of adults in North America and remains incurable. Recent discovery of targeted oral kinase inhibitors of the B cell receptor signaling pathway is transforming the therapy of CLL, but challenges remain. Individually none of these drugs is curative, and approximately 30% of patients discontinue for toxicity despite good disease response. The PI3K p110? inhibitors include the first generation FDA approved drug idelalisib, as well as duvelisib, a PI3K ? and ? inhibitor currently in registration trials. The PI3K? inhibitors have a pattern of toxicity that includes hepatitis, colitis and pneumonitis. We have two ongoing clinical trials in CLL patients, one idelalisib based and one duvelisib based, in which we have described these toxicities to be immune-mediated. We have further shown that this toxicity associates with a decrease in T regulatory cells, which could also enhance efficacy and decrease risk of solid tumors in these patients. In this grant in Specific Aim 1 we propose to deepen these observations, characterizing the T regulatory and T effector landscape of CLL during therapy with PI3K? inhibitors in relation to observed toxicities, with the goal of identifying clinical and immunologic predictors of toxicity and efficacy. We will further perform in vitro functional assays to assess the impact of PI3K? inhibition on the suppressive function of T regulatory cells, the activation capacity of T effector cells and the in vitro differentiation potential of nave CD4+ T cells. In Specific Aim 2 we will assess the impact of PI3K? knockdown on the E?-TCL1 mouse model of CLL in terms of disease development, autoimmune toxicity and T cell milieu. These experiments will focus on the mouse kinase dead genetic model but will also assess pharmacologic inhibitors. Aims 1 and 2 will therefore elucidate the fundamental biology of PI3K? selective inhibition in humans and mice and enable us to identify predictors of toxicity and efficacy that will optimize patient selection for these drugs. In Specific Aim 3, we will characterize the mechanisms of sensitivity and resistance to PI3K? inhibition. Our preliminary data implicate genetic and/or biochemical upregulation of the ERK pathway in patients who are or become resistant to PI3K? inhibition. We therefore propose to perform direct mechanistic studies of the impact of ERK pathway activation on PI3K? resistance in vitro. We will also perform additional discovery by studying patients enrolled on these two prospective clinical trials at baseline, during remission and at time of acquired resistance, including genomic, transcriptomic and signaling evaluation. This previously untreated uniform patient population represents an ideal homogeneous cohort that will allow us to understand these mechanisms much more effectively than a heterogeneous relapsed cohort. This project will therefore represent a major advance in the understanding of the biology of PI3K? inhibition in humans, in terms of its significant immunologic effects and mechanisms of resistance, all of which will allow optimal patient selection for this therapy and may even lead to expanded use of PI3K? inhibition in other cancers, based on its immunoregulatory function.